The broad, long term goals of this grant application are to understand the microbial factors and the molecular and cellular host responses to pathogens that are significant causes of infections in the anterior eye. These insights should lead to prophylactic and therapeutic strategies that can be clinically assessed for preventing or treating these serious infections. Specifically, this proposal will focus on infections caused by Pseudomonas aeruginosa and the plethora of pathogens causing keratitis and conjunctivitis that express the broadly conserved surface polysaccharide poly-N-acetyl glucosamine (PNAG). PNAG is not expressed by P. aeruginosa but is expressed by most major gram-positive and gram-negative bacterial organisms associated with eye infections including Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae, S. pyogenes, Hemophilus influenzae and Neisseria gonorrhoeae as well as by serious fungal causes of keratitis including Fusarium spp., Aspergillus niger and Candida albicans. For P. aeruginosa, we have found that this microbe enters corneal epithelial cells both in vitro and during experimental infection of the scratched-injured mouse eye via lipid rafts containing the cystic fibrosis transmembrane conductance regulator (CFTR), and that a large proportion of the bacterial cells are intracellular. The bacteril ligand for CFTR is the outer core oligosaccharide of the cell surface lipopolysaccharide. In the currently-funded proposal, we further defined some of the molecular and cellular factors activated by the P. aeruginosa-CFTR interaction in the cornea leading to destructive inflammatory responses and also analyzed in depth the ability of antibody to P. aeruginosa to prevent pathology and infection in keratitis. We initiated a rigorous study of virulence and immunity to S. aureus corneal infection including the subset that express the Panton-Valentine Leukocidin (PVL) and established the potential for immunotherapy against the PNAG surface polysaccharide to prevent or treat of S. aureus keratitis. In the ensuing years we will extend our long term goals by further analyzing the ability of antibody-mediated immunity to PNAG-expressing organisms to prevent anterior eye infections using models of keratitis and conjunctivitis, one occurring in an avascular tissue and the other in a vascularized ocular tissue. These contrasting settings will allow us to investigate, compare and contrast the host cellular co-factors needed for effective immunity which can be conducted using transgenic and germ-free mice lacking matured immune systems. Additionally, to continue our investigations into the pathogenesis of P. aeruginosa corneal infection we will use high- throughput sequencing analysis of a saturated transposon (Tn) bank of P. aeruginosa that we have recently established as a major approach to the study of the host-pathogen interaction to investigate bacterial and host factors involved in the pathogenesis of keratitis. Accomplishing these goals will provide the pre-clinical justification for testing of immunotherapies to PNAG in human ocular infections and extend the insight into the pathogenesis of P. aeruginosa keratitis to a heretofore unachieved level of precision and detail.
Eye infections are the leading cause of blindness worldwide and thus prevention and treatment with new therapies based on insights into the means used by microbes to cause eye infections should enhance the medical community's ability to prevent blindness. One component of this project will study vaccine-based approaches in mice for preventing or treating a broad range of infectious eye disease by targeting a conserved component found on the surface of many infectious agents. These results will justify testing of the vaccine in humans. Another approach uses modern cutting edge DNA sequencing technology to understand how the leading cause of contact lens infections is able to do so in the eye in order to gain a more detailed and precise insight into the infectious nature of this microbe.
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